Two of the most important problems in cell-based treatments of muscular and neurological diseases and injuries are: i) the regulation of cell proliferation and differentiation, and ii) the control of cell migration. For example, in human myoblast transplant trials for treatment of muscular dystrophies, major problems were the lack of proliferation, migration, and fusion of transplanted cells with existing muscle tissue. In repair of nerve damage, suppressing astrocyte proliferation and promoting neuroblast proliferation, along with guidance of neurite extensions are key issues. Although these cellular behaviors are critically depend on nanoscale adhesive cues in the extracellular matrix (ECM), there is currently a lack of understanding of what these crucial nanostructures are and how dynamic changes in those structures determining cell function. This lack of understanding is due, at least in part, to the lack of efficient, versatile, and convenient methods to engineer the ECM at the nanoscale across biologically relevant areas of micrometers, millimeters, and larger. The specific aims of this proposal are: i) understand and optimize a technology, called nanocrack patterning, to generate nanoengineered substrates with ECM molecule nanolines of defined widths, lengths, spacings, and orientations, ii) test the hypothesis that stretch-induced, nanoscale substrate reconfiguration can contribute to proliferation and lineage determination of myoblasts and neuroblasts, and iii) perform feasibility studies for discovery-driven research on cellular pathfinding where a microarray of criss-crossing nanopatterns of ECM molecules will be fabricated to rapidly profile cellular spreading/migrating preferences. The initial proof-of-concept studies will use C2C12 myoblasts and N27 neuronal precursor cells. Mesenchymal stems cells and primary myoblasts will be studied in the future. Although the biological problem to be addressed in this proposal is limited to myocyte and neuron behavior, the nanobiomaterials developed will be useful for addressing a much broader range of biological questions. The nanocrack patterning technique that will be developed uses nanoscale fracture mechanics and has the advantages of: (i) rapid nanopatterning over large areas (up to square centimeters and larger), (ii) nanopatterning over 3D substrates and inside microfluidic channels, (iii) generation of nanopattems consisting of multiple types of molecules on the same substrate, and (iv) stretch-induced in situ adjustment of the widths of ECM molecule nanolines generated.